Preparation of blood fraction for use in rh testing procedures



PREPARATION OF BLOOD FRACTION FOR USE IN Rh TESTING PRGCEDURES Alfred B.Kupferberg, Somerville, and Heron 0. Singher,

Plainfield, N. 3., assignors to Ortho Pharmaceutical Corporation, acorporation of New Jersey N Drawing. Application .iuly 22, 1954, SerialNo. 445,192

6 Claims. (Cl. 167-845) This invention relates to a process for thefractionation of mammalian serum and relates particularly to a processfor preparing a serum fraction containing albumin in a major amount andglobulin in a minor amount, said serum fraction being suitable for usein demonstrating the presence in blood of an Rh antibody known variouslyas incomplete antibody, blocking antibody, albumin agglutinin, andagglutinoid.

In 1939, Levine and Stetson, J. A. M. A., 113: 126, 1939 reported atransfusion reaction in a woman who delivered a macerated fetus andwhose serum contained agglutinins. In 1940, Landsteiner and Wiener,Proc. Exper. Biol. and Med. 43: 223, 1940, reported the preparation ofan anti-serum against the red cells of the rhesus monkey and found thatthis anti-Rhesus serum, when properly absorbed, agglutinated the bloodof approximately 85% of the members of the white race that were tested.They labelled the antigen in the human bloods that reacted with theanti-Rhesus serum the Rb factor. Human bloods containing the Rb factorwere termed Rh-positive. It was shown that Levine and Stetson, whoreported the earlier transfusion reaction, were dealing with a case ofRh immunization and that the unnamed property was the property laternamed the Rh factor by Landsteiner and Wiener. Levine, Katzin, andBurnharn, I. A. M. A., 116: 825, 1941, reported that intragroupincompatibility could be produced by immunization of an Rh-negativemother by an Rh-positive fetus. These investigators demonstrated at thesame time that Rh immunization explained the majority of cases oferythroblastosis fetalis.

It was at first thought that the Rh types could be explained on thebasis of the presence or absence of the Rh factor, a single positivefactor. Later it was discovered that the problem was much more complexand at the present time at least eight Rh subtypes are demon:

agglutinins, which appear in the blood of certain Rh- United StatesPatent. 9

serum was tested against a 1% or 2% suspension of Rhpositive cells in0.85% sodium chloride. It was demonstrated that these women were alsoimmunized against the Rh factor even though Rh antibodies were notdemonstrated when the serum was tested against Rh-positive cellssuspended in salt solution. I

it was later demonstrated that the serum of those women had the capacityto prevent the action of known anti-Rh agglutinins against Rh-positivecells suspended in salt solution, and it was assumed that their serumpossessed antibodies capable of nullifying the effect of ordinaryanti-Rh agglutinins on Rh-positive cells suspended in saline. body,blocking antibody, albumin agglutinin, and agglutinoid.

Hill, Reid, and Haberman, Texas State Journal of Medicine, 477-481 1949,concluded that the antibodyantigen reactions are reversible and thatthere is competi tion between the different varieties of antibodieswhich explains various antibody patterns observed clinically.

These investigators employed the term agglutinoid to designate the Rhantibody found in the euglobulin associ ated with the albumin fractionwhich saturates the antigen and blocks the action of the salineagglutinin to distinguish it from the antibodies which theseinvestigators have designated cryptagglutinoids which do not block, donot agglutinate saline suspensions, and show chemical differences tothese two antibodies. These investigators postulate that the reaction ofantibodies with antigen is reversible and that union of antigen andantibody occurs much more readilythandisassociation. The three anti:

- in reversible reactions with the antigen and following the negativeindividualsas a result of immunization by transwas later discovered thatthere were antibodies other than the saline-active, anti-Rh agglutininsresponsible for immunization. It was noted that a large proportion ofthe Rh-negative women who bore erythroblastotic children did not showanti-Rh agglutinins in their serum when the laws of mass action.Fractionation studies of these in vestigators furnished further evidencefor these three orders of Rh antibodies and it has also beendemonstrated that there are significant chemical differences between theagglutinins (saline agglutinins), and agglutino-ids (albuminagglutinins), and cryptagglutinoids. I

A test for demonstrating the presence of the incom- 1 plete antibody wasdeveloped by Diamond and Abelson, J. Lab. and Clin. Med, 30: 204, 1945,which demonstrated this antibody produced agglutination of Rh-positivecells.

In the test, one drop of the serum containing the incomplete antibodywas mixed with two drops of whole oxa-' lated or citrated Rh-positiveblood and spread thinly on a warm slide, and this resulted in clumpingof the Rhpositive cells. be demonstrated by testing the serum containingthe incomplete antibody against a 50% suspension of Rh-positive cellssuspended in serum. It was originally thought that the heavy cellsuspension was a factor which permitted such a serum to clump Rh-positvecells, but it was later concluded that the serum or plasma in which thecells were suspended was the determining factor. Itwas still later shownthat the same effect is possible when incomplete antibody is testedagainst a 2% suspension of Rh-positive cells suspended in a 20% to 30%solution of human or bovine albumin, acacia, globin and many othermacromolecular substances.

saline are incubated for one hour at 37 C. and centri- Pateut ed Sept.4, 1956;

This antibody has been called incomplete anti- The same results wereobtained and could The test to produce agglu-* tination by incompleteantibodies was later modified and t in this modification, the serum andRh-positive cells in euglobulins.

3 c a fuged. The supernatant saline is removed and serum or othermacromolecular'substance added-followed by resuspension of the cells.The mixture is again centrifuged and observed for clumping In thistest,-the Rh-po'sitive cells in saline absorb the incomplete antibodybut do not clump until the macromclecularisolution is added.

MCCullOch, Nature 165 :276277, 1950, reports on therequirements. forbovine albumin used in demonstrating incomplete Rh antibodies bysuspending the reacting cells in. a concentrated albumin solutionaccording to thetest described by Diamond, I. Clininvest, 24: 793,

1945. Prior to the report of'McCulloch, variation in the efficiency ofdifierent preparations of albumin suggested that the activating proteinwas not albumin but; some contaminating protein associated with it;Kerwi'clc and MacK'ay; 'Pro First International Congress of Biochem'-istry', l9 49greport that solutions of betaand gammaglobulin prepa redby the ether fractionation of human plasma were inactive but that anactive fraction could bejprepared from the supe'rnatant left aftertheir'precipitation which was-soluble in 0.9% saline at pH 5.0. a Thissoluble' fraction consisted of albumin 38%; alpha-globulin; 33 .495;beta-globulin, 19.7%; and gamma-globulin, 8.9%; Y Albumin, separatedelectrophoretically from this preparation'and concentratedbyfreeze-drying, showed no activity, butat a comparable proteinconcentration, the

' globulinsfwere highly active. McCulloch concluded-from these reportedresults that since the albumin was consistentl'y' ne gative, and noactivitycould be found in' betaand gamma-globulim the activity appearedto be associ atedwith alpha-globulin. 'This author suggested thatalpha-globulin is responsible for the demonstration of incomplete-Rhantibodies in the Diamond test.

Reid andlones; Amer. J. CHE. Pathology, 19: -15,

1949', described a 'pro'cess for the production of therapeuticfractionsof human bloodserum by the removal of salt with'ion exchange resins." Inthe process of these investigators, cation and anion exchange resinswere employed' alternately for removing salts from blood serum orplasma, while keeping the H between 6 and 8; As

salt' concentration progressively decreased, the globu- *linsprecipitated out until the solution was essentially saltfree and at thistime only euglobulins, albumin, hemogl'obin; and other impurities, wereleftin the solution. Precipitated globulins were removed and the pH wasbroughtdown to 5' which resulted in precipitation of the The euglobulinfraction contains the alphaglobulin components suggested byM'cCulloch tobe ne'cessary for demonstra'tion of incomplete-Rh ant-ibodiesfn the-Diamondtest, as well as beta-globulins. The precipitated euglobulinswere removed and a solution of albumin containing" the hemoglobin whichwas in the original plasma remained. Sodium caprylate was addedto0105'mol'a'rity of the albumin solution and-the pH" was ad justecb to 7.4;The caprylate stabilized the albumin so that-the solution could bepasteurized. The pH of the solution was adjusted to 5.0 and thisresulted in denaturetion oftthe-hemoglobin' which was thenquantitatively removed by centrifugation. The solution was pasteurizedandithelcaprylate" and pyrogeniccontaminants were -re'- moved. by"passage of the solution through successive columns. ofia stablecationzexch'ange resin. The albumin solution was desiccated undervacuumfrom. the: frozen alpha-globulins are precipitated.

. prevents denaturation by "heating of'the alpha-globulins 7 Heating tothis temperaturesubstantially *denatures g'lobu'lins present in the;solution; other than the active portions of "the :alpha'- stateand'bythismeans could be safely" carried to any concentration desired; Theprocess of these investigators; produced-an undenatured. albumin:suitable for clinical"v use andfreefromglobulin fractions.

It is an object of this invention to prepare a: serumfraction; suitablefor use indemonstrating. incomplete Rh antibodies in a method describedby Diamond.

, solution.

It isianother object of. thisiinv'ention to prepare 'a; serum fractionsuitable for use in demonstrating incomplete Rh antibodies 'bysuspending the reacting" cells? in a concentratedsolution of theserumfraction. v

isstillanotlienobject of this, invention; to. prepare a serumfractiomsuitable for use in the method of demonstrating incomplete Rhantibodies of Diamond in which the reacting cells are suspended'inconcentrated serum fraction, which fraction. contains albuminandalphaglobulins.

It has now been discovered that a serum fraction containing albumin andalpha globulins which is suitable for use in the Diamond test may beproduced 'from mammalian blood serum by a process in which salts areremoved from the serum with ion exchange resins toprecipitatesalt-soluble euglobulins and, after removing the precipitate, globulinsin" the serum are heat denatured'in the presence of a salt .ofan organicacid Without denaturation of albumin andalpha globu lins, andsubsequently precipitated by an acid 'adjustmentof the pH ofthe solu-.

tion and removed.

In the process of thisinvention, serum is passed through a a resincolumn containingv both cation and anion exchange'resins in a proportionsuch that a solution having. 7

a pH Within the range of from 6 to 8, and preferably neutral, resultsonpassage through the column of ani V Cation exchange resins having activephenolic, carboxyl or sulfonic groups which acidic, basic, or neutralsolution.

are saturated with hydrogen ions such as phenol methylene' sulfonic'acid resin, nuclear sulfonic acid resin, and;

polymerized acrylic acid resin and anion exchange resins having activeamine groups such as a polymerresulting. I from nitration andsubsequent-reduction'of a-tstyrenew diviulylbenzene .copolymer aresuitable for use in the process of the invention.. As the resinsremovethe'salts from :the'serum, the salt-soluble globulins, also referredtoas euglobulins, are precipitated and subsequently reg movedby'appropriate means such as centrifugation. An alkali metal salt of afatty acid having 4 toflfllca'rbon atoms is then added to the'solutionto bring its'molarity f a with respect thereto to at least about 0.15.An alkali metal salt of a fatty acid such as butyric, caproic andhexanoic acid and of acetyl tryptopha'ne has been found:

particularly suitable for addition to the'solution. Below about 0.15molar, heat subsequentlyv applied to the solution destroys activealpha-globulin componentsinece'ssary'.

for the test; when the molarity is greaterth'an about0.3',

The pH is then adjusted to between 6.8 and 7.4, but it ispreferre'd thatthe pHibe at 7.0. The solution is brought to a temperature ofapproximately 75 C. and maintained at'this tempera ture for' about 25 to35 minutes. The'alk'ali metalsalt and albumin present in the solution.

globulins. The solution is then cooled. to a temperature notgreater'than about 25C. and adjusted to'a pH of '515 to*5.8-, thepreferred pH being 56., Adjustment of: thepH of the solution to 5.5 to5.8. after heating results; in precipitation of substantially alldenatured gl'obu'lins,

leaving undenaturedalpha:globulins in'solutiom. If the pH is adjusted toa morealkaline reaction there is insufi icient precipitationof denaturedglobulins'. Thepre' V. I 'cipitated-denaturcdglobulins are removed byappropriate .means such as centrifugation and the'pH of thesupernata'nt'may then be adjusted to approximately7 in order thatj the end productmay besuitable for use in the Dia'.

mond test} The alkali-metal salt of thefa'ttygacid or Diamond.

I The invention is further illustratedlby the 'foflowing example, but itshould beundersto'od that the. invention" is; not limited. to

l l the specific details set' forth in the examp e. V r V t x 2,650 ml.of bovine serum were passed through a resin column consisting of amixture of a strong-base anion exchange resin, Amberlite IRA-$00 havinga total capacity of 2.3 mini-equivalents per gram, the resin being areaction product of trimethyl amine and an insoluble, crosslinkedcopolymer of styrene and divinyl benzene, which copolymer containschloroalkyl groups having the formula CTLH271C1, in which CnHZr. is analkylene group and in which n is an integer from 1 to 4, and a phenolmethylene sulfonic acid cation exchange resin, Amberlite lR-lOS having atotal capacity of 2.70 milliequivalents per gram. The proportion of thecation exchange resin and anion exchange resin in the column was suchthat a neutral solution resulted on passage through the column of anacidic, basic, or neutral solution. After the serum had passed throughthe column, 1,350 cc. of Water were passed through to Wash out any serumremaining therein. The total efiiuent from the column containedprecipitated globulin which was removed from the solution bycentrifugation. 90.88 g. of sodium capryllate was added to the 3,650 cc.of solution remaining after removal of precipitated globulin and thisresulted in a 0.15 molar solution of capryllate. The pH of the solutionwas adjusted to 7.0 with sodium hydroxide and the solution was thenslowly heated over a period of 145 minutes to approximately 75 C. andheld at this temperature for 30 minutes. The solution was cooled tosubstantially room temperature and the pH adjusted to 5.6 at which timea precipitation of denatured globulin resulted which was removed bycentrifugation. The resulting supernatant had a volume of 2,400 cc. andafter adjustment of the pH to 7.0, the solution was dialyzed in order toremove salts contained therein, and freeze-dried. The yield of serumfraction was grams per liter of original serum.

The following example illustrates the use of the serum fraction obtainedby the above procedure in the test procedure of Diamond, a solution ofthe serum fraction being substituted for the solution of human or bovinealbumin, acacia, globin or other macromolecular substance used in theDiamond procedure.

A blood sample suspected of containing the blocking antibody was allowedto clot and the clotted sample was centrifuged. Two drops of the serumobtained by centrifugation were placed in a test tube and to this wasadded one drop of a physiological salt solution containing in solution22% by weight of the serum fraction prepared according to the exampleabove and 2% by weight of Rh positive blood cells were suspended in themixture. The mixture was incubated for one hour in a water bathmaintained at 37 C., removed from the water bath; centrifuged at 1000revolutions per minute for one minute, removed from the centrifuge,agitated gently and examined microscopically. The red blood cellsexhibited the phenomenon known as clumping, thus demonstrating thepresence in the test serum of the blocking antibody.

Since certain changes may be made in the above process and differentembodiments of the invention could be made without departing from itsscope, it is intended that all matter contained in the above descriptionshould be interpreted as illustrative and not in a limiting sense.

This application is a continuation in part of our application Serial No.273,782, filed February 27, 1952, now abandoned.

What is claimed is:

1. The process of preparing blood serum proteins suitable for use indemonstrating incomplete Rh antibodies by suspending reacting bloodcells in a solution of the blood serum proteins which comprises passingmammalian blood serum through a column containing both cation and anionexchange resins in a proportion such that a solution having a pH withinthe range of from 6 to 8 results on passage through the column of asolution selected from the class consisting of acidic, basic, andneutral solutions, whereby a precipitate of euglobuhas is formed in thesolution; removing the precipitate; adding to the solution an alkalimetal salt of an organic acid selected from the group consisting offatty acids having from 4 to 10 carbon atoms and acetyl tryptophane inan amount sufiicient to bring the molarity of the solution with respectthereto to at least about 0.15; adjusting the pH of the solution to6.8-7.4; heating the solution to approximately C. and maintaining thetemperature of the solution at approximately this temperature for aperiod of from 25 to 35 minutes; cooling the solution to a temperaturenot greater than about 25 C.; adjusting the pH of the solution to5.5-5.8, whereby a precipitate of denatured globulins is formed;removing the precipitated denatured globulins; and removing from thesolution the alkali metal salt of the organic acid.

2. A process according to claim 1 in which the alkali metal salt of anorganic acid is sodium capryllate.

3. A process according to claim 1 in which the cation exchange resin hasan active group selected from the class consisting of phenolic,carboxyl, and sulfonic groups and in which the anion exchange resin hasan active amine group.

4. A process according to claim 1 in which the cation exchange resin isa phenol methylene sulfonic acid resin and in which the anion exchangeresin is a polymer resulting from nitration and subsequent reduction ofa styrene-divinylbenzene copolymer.

5. The process of preparing bovine blood serum proteins suitable for usein demonstrating incomplete Rh antibodies by suspending reacting bloodcells in a solution of the blood serum proteins which comprises passingbovine serum through a column containing both cation and anion exchangeresins in a proportion such that a solution having a pH within the rangeof from 6 to 8 results on passage through the column of a solutionselected from the class consisting of acidic, basic, and neutralsolutions, whereby a precipitate of euglobulins is formed in thesolution; removing the precipitate; adding to the solution an alkalimetal salt of an organic acid selected from the group consisting offatty acids having from 4 to 10 carbon atoms and acetyl tryptophane inan amount sufiicient to bring the molarity of the solution with respectthereto to at least about 0.15; adjusting the pH of the solution to6.8-7.4; heating the solution to approximately 75 C. and maintaining thetemperature of the solution at approximately this temperature for aperiod of from 25 to 35 minutes; cooling the solution to a temperaturenot greater than about 25 C.; adjusting the pH of the solution to5.5-5.8, whereby a precipitate of denatured globulins is formed;removing the precipitated denatured globulins; and removing from thesolution the alkali metal salt of the organic acid.

6. A process according to claim 5 in which the alkali metal salt of anorganic acid is sodium capryllate.

References Cited in the file of this patent McCulloch: Nature, vol. 165,pp. 276-277, Feb. 18, 1950.

Reid et al.: Am. J. Clin. PathoL, vol 19, No. 1, pp. 10-15, January1949.

Greenberg: Amino Acids and Proteins, 1st ed. (1951), pp. 279-287.

Cohn et al.: I. A. C. S., vol. 68, March 1946, p. 459- 475 (p. 460relied upon).

1. THE PROCESS OF PREPARING BLOOD SERUM PROTEINS SUITABLE FOR USE INDEMONSTRATING INCOMPLETE RH ANTIBODIES BY SUSPENDING REACTING BLOODCELLS IN A SOLUTION OF THE BLOOD SERUM PROTEINS WHICH COMPRISES PASSINGMAMMALIAN BLOOD SERUM THROUGH A COLUMN CONTAINING BOTH CATION AND ANIONEXCHANGE RESINS IN A PROPORTION SUCH THAT A SOLUTION HAVING A PH WITHINTHE RANGE OF FROM 6 TO 8 RESULTS ON PASSAGE THROUGH THE COLUMN OF ASOLUTION SELECTED FROM THE CLASS CONSISTING OF ACIDIC, BASIC, ANDNEUTRAL SOLUTIONS, WHEREBY A PRECIPITATE OF EUGLOBULINS IS FORMED IN THESOLUTION; REMOVING THE PRECIPITATE; ADDING TO THE SOLUTION AN ALKALIMETAL SALT OF AN ORGANIC ACID SELECTED FROM THE GROUP CONSISTING OFFATTY ACIDS HAVING FROM 4 TO 10 CARBON ATOMS AND ACETYL TRYPTOPHANE INAN AMOUNT SUFFICIENT TO BRING THE MOLARITY OF THE SOLUTION WITH RESPECTTHERETO TO AT LEAST ABOUT 0.15; ADJUSTING THE PH OF THE SOLUTION TO6.8-7.4; HEATING THE SOLUTION TO APPROXIMATELY 75* C. AND MAINTAININGTHE TEMPERTURE OF THE SOLUTION AT APPROXIMATELY THIS TEMPERATURE FOR APERIOD OF FROM 25 TO 35 MINUTES; COOLING THE SOLUTION TO A TEMPERATURENOT GREATER THAN ABOUT 25* C.; ADJUSTING THE PH OF THE SOLUTION TO5.5-5.8, WHEREBY A PRECIPITATE OF DENATURED GLOBULINS IS FORMED;REMOVING THE PRECIPITATED DENATURED GLOBULINS; AND REMOVING FROM THESOLUTION THE ALKALI METAL SALT OF THE ORGANIC ACID.